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Abstract

Introduction

Systemic lupus erythematosus (SLE) patients have lower bone mineral density and increased
fracture risk when compared with healthy individuals, due to distinct factors and
mechanisms. Bone remodeling is a tightly orchestrated process dependent on several
factors, including the balance between receptor activator of nuclear factor κB ligand
(RANKL) and osteoprotegerin (OPG).

Our aim was to assess serum OPG and soluble RANKL (sRANKL) levels as well as sRANKL/OPG
ratio in female SLE patients and compare it with female controls.

Methods

We have evaluated 103 SLE patients and 114 healthy controls, all Caucasian females.
All participants underwent a clinical and laboratory evaluation. sRANKL and OPG were
quantified in serum by ELISA based methods. sRANKL, OPG and sRANKL/OPG ratio levels
were compared between SLE patients and age, sex and race matched healthy controls.
For SLE patients, a multivariate analysis was performed, to find the possible predictors
of the changes in sRANKL, OPG and sRANKL/OPG ratio levels.

Results

Although sRANKL levels did not differ between the two groups, serum OPG was lower
in SLE patients (P < 0.001). This led to an increased sRANKL/OPG ratio (P = 0.010) in the patients' group.

The multivariate analysis was performed considering age and other clinical and laboratorial
potential confounders for these variations in the SLE patients group. We have showed
that age (P = 0.001) and levels of anti-Sm antibodies (P = 0.016) were independent predictors of sRANKL/OPG ratio variations in SLE patients.
No relationship with therapy or disease activity measured by SLEDAI2K was found.

Conclusions

These results are suggestive of increased osteoclastic stimuli driven by the SLE disease
mechanisms.

Keywords:

Introduction

Systemic lupus erythematosus (SLE) is a chronic, multisystemic disease of unknown
etiology characterized by chronic inflammation and damage to various organs and systems
due to the production of autoreactive cells and antibodies [1-3].

SLE patients have lower bone mineral density (BMD) when compared with healthy individuals
and are at increased risk of fracture [4-7]. Although corticosteroid exposure is a major contributor to bone loss in SLE [4,5,8]. disease activity and associated co-morbidities may contribute to this process [5,8]. In addition, vitamin D deficiency is a common finding among SLE patients, further
contributing to impaired bone health [5].

Bone remodeling is a tightly orchestrated process in which osteoclasts attach to the
bone surface and remove bone. After resorption, osteoblasts migrate into the lacunae
and produce new bone, which then mineralizes [3,9]. Expression of the receptor activator of nuclear factor κB ligand (RANKL) by osteoblasts
is essential to osteoclastogenesis. Osteoprotegerin (OPG) is a soluble receptor for
RANKL that prevents RANK/RANKL interaction. Therefore, the RANKL/OPG ratio is critical
for the control of bone resorption [10-12]. Increased RANKL/OPG ratio has been described in autoimmune diseases, such as rheumatoid
arthritis, and was associated with an increased bone loss [7,13,14]. Taking these arguments into consideration, we have hypothesized that a RANKL/OPG
imbalance is also present in SLE patients.

In the present work we aimed to assess the RANKL/OPG balance in SLE patients by quantifying
serum OPG and sRANKL levels and their ratio in SLE patients and healthy controls.
Additionally, in SLE patients we have looked at predictors of serum levels of these
proteins and having as covariates disease features, co-morbidities and medications.

Materials and methods

Patients

Consecutive Portuguese Caucasian SLE women were recruited from the rheumatology outpatient
clinics of Hospital de Santa Maria, Lisbon and Hospital Garcia de Orta, Almada, Portugal.
All enrolled patients fulfilled the classification criteria of the American College
of Rheumatology for SLE (1997) and had normal renal function defined as serum creatinine
< 1.5 mg/dl. A control group matched to age, sex and race was also recruited, and
was composed of healthy Caucasian female volunteers, who had not been diagnosed with
SLE, nor had any inflammatory or bone disease and were not receiving corticosteroids
or other medications known to interfere with bone metabolism. In this study, 103 SLE
patients and 114 healthy controls were enrolled. This study was approved by the local
Ethics Committees and all participants signed a written informed consent.

Serum was obtained by blood centrifugation at 1,250 g, 10', at 4°C and then preserved
at -80°C until use for sRANKL and OPG quantification.

sRANKL quantification was performed using the ampli sRANKL human ELISA (Immunodiagnostic
Systems, Boldon, UK). OPG was quantified using the Bender MedSystems (Vienna, Austria)
bead-based assay for quantitative detection of soluble human analytes by flow cytometry.
Both protocols were performed according to the manufacturer's instructions.

Statistical analysis

For statistical purposes samples undetectable or below the limit of detection (LOD)
were considered to have the lower LOD value supplied by the manufacturer. Results
were reported as means (standard deviation), medians (interquartile range) for continuous
or proportions for categoric variables. sRANKL and OPG levels and sRANKL/OPG ratio
were compared between SLE patients and healthy controls groups using the non-parametric
Mann-Whitney test.

Subsequently, the impact of demographic parameters, clinical features, therapeutics
and disease characteristics on these proteins and their ratio was investigated for
SLE patients using univariate followed by multivariate linear regression analyses.
All variables related to the studied outcome in univariate analyses at a P-value < 0.05 were considered potential predictors and entered in multivariate linear
regression models. The selection of covariates was stepwise by backward selection,
according to the level of significance. Before performing regression analysis, sRANKL,
OPG, and sRANKL/OPG ratio were logarithmically transformed for approximation to normality
and to approximate the residuals to the normality in multiple linear regressions.

Statistical calculations were performed using Statistical Package for the Social Sciences
(SPSS) Statistics Software, v.15.0 (SPSS Inc., Chicago, USA) and a two-tailed P-value < 0.05 was selected as significant.

Results

A total of 103 SLE patients and 114 healthy controls with comparable baseline demographic
characteristics and co-morbidities were studied (Table 1). SLE patients had a mean age at disease diagnosis of 35.6 ± 14.4 (range 9.0 to 80.0)
years, 8.2 ± 6.6 (range 0.2 to 34.2) years of disease duration, a mean SLEDAI2K of
3.5 ± 4.5 (range 0 to 21) and a SLICC damage score of 0.8 ± 1.4 (range 0 to 8). A
total of 60.2% of the patients were currently receiving corticosteroids in a mean
daily dose of 12.7 mg.

No significant differences were found between the two groups regarding sRANKL concentration.
However, a statistically significant lower value was found for OPG levels in SLE patients
(P < 0.001) compared to healthy controls. Consequently, an increase in sRANKL/OPG ratio
(P = 0.010) was found in SLE patients compared to the healthy control group (Table 2).

The same reasoning was applied to the analysis of the sRANKL/OPG ratio in SLE patients.
In the univariate analysis age, BMI, menopausal status, lipid-lowering therapy, age
at disease onset, the presence of malar rash, pleuritis and the levels of anti-dsDNA
and anti-Sm antibodies came out as possible predictors for changes in the ratio values.
After multivariate analysis age (β = -0.326, 95% CI -0.055 to -0.015; P = 0.001) and levels of anti-Sm antibodies (β = 0.229, 95% CI 0.006 to 0.053; P = 0.016) were independently associated with sRANKL/OPG ratio levels in SLE patients.

We found no relationship between sRANKL/OPG ratio and the inflammatory parameters
ESR and CRP. In addition, we found no association between the ratio and concomitant
medications, such as methotrexate, cyclophosphamide, mycophenolate mofetil or azathioprine.
Furthermore, there was also no correlation with corticosteroids (use or actual dose)
or disease activity measured by the SLEDAI2K with this ratio.

Although the studied SLE patients presented a wide range of disease duration, this
variable did not come out as a predictor of sRANKL, OPG or SRANKL/OPG levels.

Discussion

The present work provides evidence of increased pro-osteoclastogenic stimuli in SLE
women as a result of decreased serum OPG levels and increased sRANKL/OPG ratio.

OPG serum levels were lower in SLE patients than in controls and these levels were
negatively associated with anti-dsDNA levels, independently from the contribution
of multiple confounders. Raised anti-dsDNA levels are associated with active disease,
suggesting that patients with active SLE might be more exposed to the effect of RANKL/RANK
interaction as a consequence of diminished OPG levels. We have also found a positive
association between serum OPG levels and diabetes mellitus, which is in accordance
with previous results [18,19]. Gannagé-Yared and colleagues found an inverse correlation between OPG and triglycerides
levels, in a nondiabetic, elderly Lebanese male population [20]. Although in a different population, this relation was not confirmed by our study,
since we have found a positive relation between serum OPG and triglycerides.

Serum OPG levels have been scarcely analyzed in the context of SLE. There is a single
study reporting higher serum OPG levels in SLE patients, and this relation is even
greater in patients with antiphospholipid syndrome, as OPG levels were related to
the presence of antiphospholipid antibodies [21]. This relation between OPG and these antibodies was not confirmed by us (data not
shown).

On the other hand, urinary OPG levels have been described to be raised in lupus nephritis
and correlated with renal disease activity and anti-dsDNA levels [22,23]. However, at this moment it is not clear how these results can be compared with ours
as the relationship between serum and urinary OPG levels is unknown.

We have found sRANKL levels to be similar between SLE and healthy control women, but
the sRANKL/OPG ratio was increased in SLE patients as compared to controls at the
cost of elevated serum OPG levels in SLE. Interestingly, malar rash and elevated levels
of anti-Sm autoantibodies, often present in active disease, were associated with sRANKL
serum levels. Moreover, in multivariate analysis levels of anti-Sm antibodies were
positively associated with sRANKL/OPG ratio. Studies based on sRANKL are sometimes
limited by the high percentage of patients that have undetectable circulating levels,
due to the fact that the majority of RANKL is membrane bound. Nevertheless, half of
our patients had detectable levels of sRANKL and, importantly, it was possible to
determine serum OPG levels in all individuals.

Despite the fact that there are no previous references in the literature to the possible
effect of SLE on sRANKL and sRANKL/OPG ratio, an imbalance of this ratio has been
described in autoimmune diseases, such as rheumatoid arthritis [13,24]. This finding may be of clinical relevance as the increase of the sRANKL/OPG ratio
has been related with increased bone loss in immune mediated inflammatory diseases
[12,25]. The independent association of the sRANKL/OPG ratio with anti-Sm autoantibodies
and the absence of association with corticosteroid use or dose are particularly relevant,
as they are suggestive that SLE per se might be important in accelerating osteoclastogenesis and consequently, bone loss.

Conclusions

In summary, we have shown reduced OPG levels and consequently a raised sRANKL/OPG
ratio in female SLE patients as compared to healthy controls. An association between
anti-dsDNA and OPG levels and between anti-Sm and sRANKL levels and sRANKL/OPG ratio
were also observed in SLE patients. Taken together, these observations are suggestive
of increased osteoclastic stimuli driven by SLE disease mechanisms.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

DCF carried out laboratorial protein determinations and participated in the design
of the study, statistical analysis and manuscript elaboration. MJS performed clinical
evaluation of the patients and participated in the design of study, statistical analysis
and manuscript elaboration. IPP helped on laboratorial protein determinations and
on manuscript revision. JEF participated in the design of the study and on manuscript
revision. HC participated in the design of the study and on manuscript and statistical
analysis revision. All authors read and approved the final manuscript for publication.

Acknowledgements

This work was supported by a grant from Fundação para a Ciência e a Tecnologia, Portugal
(PIC/IC/82920/2007) and by an unrestricted grant from Wyeth Portugal.

We wish to thank Dr. Vinagre F. from the Rheumatology Department at Hospital Garcia
de Orta for helping with patients' data collection and Dr. Caetano-Lopes J. from the
Rheumatology Research Unit at Instituto de Medicina Molecular for advice with statistical
analysis.